The project extensively relied on high-resolution all-atom molecular dynamics simulations and absolute binding free energy calculations performed using the polarizable AMOEBA force field. The study is complemented by extensive adaptive sampling simulations that are used to rationalize the different ligand binding poses through the explicit reconstruction of the ligand–protein conformation space. Machine learning predictions are also performed to predict selected compound properties. While simulations extensively use high performance computing to strongly reduce the time-to-solution, they were systematically coupled to nuclear magnetic resonance experiments to drive synthesis and for in vitro characterization of compounds. Such a study highlights the power of in silico strategies that rely on structure-based approaches for drug design and allows the protein conformational multiplicity problem to be addressed. The proposed fluorinated tetrahydroquinolines open routes for further optimization of Mpro inhibitors towards low nM affinities.We report a fast-track computationally driven discovery of new SARS-CoV-2 main protease (M The dominant binding mode of the QUB-00006-Int-07 main protease inhibitor during absolute binding free energy simulations. Despite these efforts, few potential treatments are presently available with the exception of Paxlovid, a nirmatrelvir/ritonavir combo proposed by Pfizer.7 Due to the persistence of the pandemic, it remains essential to propose new antiviral drugs. A possible strategy consists in designing small molecules to interact with one of the main proteins of the SARS-CoV-2 virus, thus blocking its activity. Among the potential targets, the main protease protein, denoted as Mpro or 3CLpro, is a primary choice8 as it has no human homolog and it is well conserved among coronaviruses,9 especially in terms of the structure of its active site, catalytic dyad, and dimer interface. Furthermore, Mpro is required to release viral proteins for particle assembly, and is thus essential to the virus replication cycle.Since December 2019, the COVID-19 global pandemic has put the entire world on edge.pro is challenging as it requires extensive resources and the success rate is notoriously low.10 Relying on in silico driven rational design could accelerate the process. In fact, it diminishes the cost by reducing the need for synthetic iterations while also providing an interpretation of the interactions occurring between the target protein and potential inhibitors.Developing a new drug targeting the viral Mpro is challenging as the protein exhibits high structural flexibility11–13 leading to high conformational complexity. Mpro is also involved in a variety of complex protein–ligand–solvent interaction networks.12,13 These challenges can be tackled using a high-resolution modeling approach12,13 going beyond rigid docking procedures (see ref. pro).It is important to note that theoretical modeling of Mpro inhibitors3,5,15–25 through joint computational and experimental approaches. In particular, a recent study by the Jorgensen group highlighted the usefulness of relative binding free energy (RBFE) computations as part of the drug design process.26Many studies have been devoted to the design of new Mpro inhibitors. In doing so, we build on our previous high-resolution Mpro molecular dynamics studies.12,13 Here, we explore more deeply some specific subpockets of the substrate binding site of the protease using absolute binding free energy (ABFE) calculations and adaptive sampling grounded on extensive molecular dynamics simulations with high-resolution polarizable force fields (PFFs). Using the GPU-accelerated module27 (GPU = Graphics Processing Unit) of the Tinker-HP molecular dynamics package28 coupled to the AMOEBA PFF,29–32 it has been shown that simulations can reach the required level of accuracy and μs timescales needed to explore the structural rearrangement and interactions profile of this flexible protein.12,13 More precisely, the modeling of Mpro necessitates the ability to evaluate at high resolution various types of key interactions including hydrogen bonds, salt bridges, π–π stacking, and specific solvation effects. Long timescales are required to achieve sufficient sampling. This is now possible by using the large number of graphics processing units (GPUs) that are presently available on supercomputers and high-performance cloud computing platforms. In this study, we combine our computationally driven strategy, using absolute binding free energy computations33–37 and unsupervised adaptive sampling,12,13 with machine learning-assisted property predictions, while conducting extensive characterization experiments including nuclear magnetic resonance (NMR), mass spectrometry (MS), and FRET-based assays to evaluate the activity of the newly designed compounds.In this paper, we present a computationally driven discovery and binding mode rationalization of new SARS-CoV-2 Mpro .In the following, we introduce our design strategy, which led to non-covalent and covalent inhibitors of M2.2.1.7L11) was used for all the MD simulations and it was prepared at physiological pH (pH = 7). This structure has a higher resolution (1.80 Å) than the PDB structure (PDB code: 6LU7) used in our previous work12 (resolution of 2.16 Å). Both structures are of the holo state in complex with covalent inhibitors, and the rotamers of the key residues at the catalytic site  are virtually identical. The protonation states of His residues were assigned based on previous work,38 where His41 and His80 are protonated at the delta carbon atom and all other His residues are epsilon-protonated, which is favorable for substrate binding.38 This is different from our previous work where His64 and His80 are protonated at the delta carbon atom and all other histidines are epsilon-protonated.12 All water molecules were retained except for those that might collide with the ligands.The protease dimer structure  system including cloud supercomputing infrastructures. Among its possibilities, Atlas has the ability to efficiently handle polarizable force field molecular dynamics simulations using a custom version of the multi-GPU module2.2.1.27 The AMOEBA polarizable force field29–32 was used to describe the full systems including the protein, ions and water. Several utilities (TinkerTools) from Tinker 8 ,45 a preconditioned conjugate gradient polarization solver (with a 10−5 convergence threshold) to solve polarization at each time step,46 hydrogen-mass repartitioning (HMR) and random initial velocities. Absolute binding free energy simulations following a protocol described in the next section were performed as well as adaptive sampling runs that are also described further in the text. Absolute binding free energy computations were both performed on the HPE Jean Zay Supercomputer  and on Amazon Web Services (AWS). All adaptive sampling computations were performed using AWS. Simulations on AWS used both p3.2x (NVIDIA V100 GPU cards) and p4d.24xlarge (NVIDIA A100 GPU cards) instances whereas computations on the Jean Zay supercomputer were powered by V100 cards.All Tinker-HP MD simulations  were performed in mixed precision to benefit from a strong acceleration of simulations using GPUs.r 8 ref.  were use2.2.2.R) and QUB-00006-Int-01(S) into the Mpro dimer structure using Autodock Vina 1.1.2.40 AutoDock Vina requires the pdbqt format for the input files of the receptor and the ligand. Therefore, using the scripts ‘prepare_receptor4.py’ (v 1.13) and ‘prepare_ligand4.py’ (v 1.10) provided by Autodock Tools,47 we generated pdbqt files corresponding to the receptor and the ligands, respectively. We set the exhaustiveness search to 100 and the num_mode option to 50.The protonation states of the ligands were calculated at a neutral pH and the hydrogen atoms were added using Chimera. Next, we docked the ligands QUB-00006-Int-01 flexible during this sampling phase. The restart strategy 51 method were used to calculate the standard binding free energy for each binding pose.33,37 There were 27 or 26 thermodynamic states for the decoupling in the complex phase or the aqueous phase. A distance restraint between two groups of atoms in the ligand and in the protein binding pocket was applied when decoupling the ligand in the complex to accelerate the convergence when the ligand is fully decoupled, and the restraint was removed at an additional step at the full interaction state. A harmonic restraint with a force constant of 15.0 kcal mol−1 Å−2 and radius of 2.0 Å was used. An analytical correction was added to the binding free energy to account for the standard state at 1.0 mol L−1 in the fully decoupled state. 10 ns simulations were performed for each thermodynamic state for the simulations of Mpro in complex with x0195, QUB-00006(S), QUB-00006(R), and QUB-00006-Int-07. For the simulations of Mpro in complex with QUB-00006-Int-01(R) and QUB-00006-Int-01(S), we ran each thermodynamic state for 20 ns. We used the BAOAB-RESPA1 integrator with a 10 fs time step and we calculated the electrostatic interactions using Ewald summation with a real space cutoff of 7 Å. van der Waals interactions were calculated using a cutoff of 12 Å with long-range correction.In order to benefit from the high-accuracy evaluation of free energies using the AMOEBA force field,2.3.S, S measured in mol L−1) and octanol/water partition coefficient (log P). To build a water solubility QSPR predictor, the AqSolDB dataset52 was used as a training set. To predict octanol/water partition coefficients (log P), the dataset from EPA's OPERA53 was used as a training set.Qubit Pharmaceuticals' Atlas internal machine learning-based QSPR module was used to predict the water solubility . The Morgan circular fingerprint55 with radius = 2 and nBits = 2048 from RDKit was also tested and the results (not shown) exhibit a similar ranking of the compounds.Tanimoto similarity3.3.1.pro56 was a generous gift from Prof. Rolf Hilgenfeld, University of Lübeck, Lübeck, Germany. Protein expression and purification were adapted from Zhang et al.56 The expression plasmid was transformed into E. coli strain BL21 (DE3) and then pre-cultured in YT medium at 37 °C (100 μg mL−1 ampicillin) overnight. The pre-culture was used to inoculate fresh YT medium supplied with an antibiotic and the cells were grown at 37 °C to an OD600 of 0.6–0.8 before induction of overexpression with 0.5 mM isopropyl-d-thiogalactoside (IPTG). After 5 h at 37 °C, cells were harvested by centrifugation  and frozen. The pellets were resuspended in buffer A  supplemented with lysozyme, DNase I and PMSF for the lysis. The lysate was clarified by centrifugation at 12 000g at 4 °C for 1 h and loaded onto a HisTrap HP column  equilibrated with 98% buffer A/2% buffer B . The column was washed with 95% buffer A/5% buffer B and then His-tagged Mpro was eluted with a linear gradient of imidazole ranging from 25 mM to 500 mM. Pooled fractions containing the target protein were subjected to buffer exchange with buffer A using a HiPrep 26/10 desalting column . Next, PreScission protease was added to remove the C-terminal His tag (20 μg of PreScission protease per mg of target protein) at 12 °C overnight. Protein solution was loaded onto a HisTrap HP column connected to a GSTrap FF column  equilibrated in buffer A to remove the GST-tagged PreScission protease, the His-tag, and the uncleaved protein. Mpro was finally purified with a Superdex 75 prep-grade 16/60  SEC column equilibrated with buffer C . Fractions containing the target protein at high purity were pooled, concentrated at 25 mg mL−1 and flash-frozen in liquid nitrogen for storage in small aliquots at −80 °C.The plasmid pGEX-6P-1 encoding SARS-CoV-2 M3.2.pro was determined by direct infusion electrospray ionization mass spectrometry (ESI-MS) on a Xevo G2-XS QTOF mass spectrometer (Waters). Samples were diluted in 50% acetonitrile with 0.1% formic acid to achieve a final 1 μM concentration of protein. The detected species displayed a mass of 33 796.64 Da, which matches very closely the value of 33 796.81 Da calculated from the theoretical full-length protein sequence (residues 1–306). To characterize the enzymatic activity of our recombinant Mpro, we adopted a FRET-based assay using the fluorogenic substrate 5-FAM-AVLQ′SGFRK(DABCYL)K (ProteoGenix) harbouring the cleavage site of SARS-CoV-2 Mpro (′ indicates the cleavage site). The fluorescence of the intact peptide is very low since the fluorophore 5-FAM and the quencher Dabcyl are in close proximity. When the substrate is cleaved by the protease, the fluorophore and the quencher are separated, increasing the fluorescence signal. Freshly unfrozen recombinant SARS-CoV-2 Mpro was used in our assays. The assay was performed by mixing 0.05 μM Mpro with different concentrations of substrate (1–128 μM) in the reaction buffer  in the final volume of 100 μL. Fluorescence intensity (Ex = 485 nm/Em = 535 nm) was monitored at 37 °C with a Victor3 microplate reader (PerkinElmer) for 50 min. A calibration curve was created by measuring multiple concentrations (from 0.001 to 5 μM) of free fluorescein in a final volume of 100 μL reaction buffer. Initial velocities were determined from the linear section of the curve, and the corresponding relative fluorescence units per unit of time (ΔRFU/s) were converted to the amount of the cleaved substrate per unit of time (μM s−1) by fitting to the calibration curve of free fluorescein. Inner-filter effect corrections were applied for the kinetic measurements according to ref. kcat/km resulted in 4819 ± 399 s−1 M−1, in line with literature data.56,58The molecular mass of the recombinant SARS-CoV-2 M3.3.59 and Saturation Transfer Difference (STD)60 experiments in the presence and in the absence of the protein. Samples contained 10 μM Mpro and 100 μM to 2 mM ligand dissolved in 150 mM NaCl, 20 mM phosphate, 5% D2O, and 4% DMSO-d6 (pH = 7.3). WaterLOGSY experiments were performed with a 180° inversion pulse applied to the water signal at 4.7 ppm using a Gaussian-shaped selective pulse of 5 ms. Each WaterLOGSY spectrum was acquired with 320 scans, a mixing time of 1.5 s and a relaxation delay of 4.5 s. STD experiments were performed with 256 scans. Selective saturation of the protein at 0.4 ppm frequency was carried out by a 2 s pulse train  included in the relaxation delay and a 30 ms spin-lock was used to reduce the broad background protein signal. The estimation of the KD was achieved by a STD titration according to a previously reported procedure and fitting the curves using OriginPro 2018 . The water suppression was achieved by the excitation sculpting pulse scheme.All the NMR screening experiments were performed with a Bruker Neo 600 MHz spectrometer, equipped with a nitrogen cooled 5 mm Prodigy CryoProbe at 298 K. The ligand binding was monitored by WaterLOGSY (wLogsy)3.4.pro was used to evaluate the ability of the compounds to inhibit its activity in vitro. In fact, inhibition of Mpro by the tested compounds results in a decrease of the fluorescence signal compared to the Mpro activity in the absence of an inhibitor. A preliminary screening was first performed at a single compound concentration to rapidly identify the ability of the compounds to inhibit Mpro activity and to rank them according to their inhibitory activity. The protein was diluted in the reaction buffer  and pipetted into a 96-well plate to a final protein concentration of 0.02 μM in a final volume of 100 μL. Each compound at the final concentration of 100 μM was incubated with Mpro for 20 minutes at room temperature. After incubation, the peptide substrate  was added to initiate the reaction which was monitored for 50 min at 37 °C. The final DMSO amount was 3.75%. Two controls were prepared for each experiment: the peptide substrate in the absence of Mpro  and the reaction mixture in the absence of the compounds . Following the preliminary screening, the most active compounds (hits) were tested at increasing concentrations  to determine the dose–response curves and calculate IC50 values fitted using GraphPad Prism 5 software. Each experiment was performed in triplicate and the results were used to calculate an average and a standard deviation.A FRET-based assay employed to test the enzymatic activity of the recombinant SARS-CoV-2 M3.5.pro  with each compound in the reaction buffer . The final mixtures had a 1 : 1 or 10 : 1 compound : protein molar ratio. Samples were incubated at room temperature for 20 min before analysis. Control experiments were performed on 10 μM solutions of Mpro in the absence of the compounds. Mass spectrometric analyses were carried out in positive ion mode by ESI-MS under denaturing conditions, i.e. water/acetonitrile 50 : 50 with 0.1% formic acid on a Q-Tof Xevo G2S . Data were processed using MassLynx V4.1 software.Samples were prepared by mixing appropriate volumes of M3.6.The detailed synthetic protocol used to prepare all molecules can be found in the ESI.‡4.pro have been identified by high-throughput crystallographic screening of this protease. Among the screened fragments, x0195  is reaching the boundaries of the hydrophobic part of the binding pocket composed of the alkyl chain of Pro168.In the co-crystal structure corresponding to M0195 see , the aro64 we noticed that x0195 was modeled in a high energy conformation and that an unusual high-energy (i.e. repulsive) contact occurs between the sulfonamide oxygen and the carbonyl oxygen of the Glu166 backbone. Additionally, the tetrahydroquinoline scaffold was not fully exploring S2 subpocket boundaries. As reported by Cannalire et al.63 and Zhang et al.,8 the volume of the S2 subpocket in SARS-CoV Mpro is very similar to that of the MERS-CoV homologue. However, the volume of S2 in SARS-CoV Mpro (252 Å3) is significantly larger than in other CoV homologues of the α-genus, such as the HCoV-NL63 Mpro (45 Å3).8,63 Therefore, exploiting this knowledge might be key to designing specific inhibitors of CoV Mpro.After comparing the available X-ray structural information with previously conducted studies on small molecule conformational preferences derived from crystal structure data,e.g. protein flexibility and binding pocket rearrangements12,13) to guide the design of better binders of the subpocket S2, we ran all-atom molecular dynamics simulations using the AMOEBA polarizable force field29–32 on Mpro (PDB code: 7L11) in complex with x0195 (PDB code: 5R81).In order to refine the available X-ray structural model and to gather more structural insights  the SO2 angle, compared with a 90° value for the same angle as reported in the crystal structure  provided in the literature62 using the Gibbs free energy equation and the experimental temperature used in the binding assays (283 K). The agreement of the computed free energy prediction with the experimental results is reasonable. Further analysis of MD simulations suggests that the tetrahydroquinoline scaffold of x0195 is sub-optimally occupying the binding pocket.We obtained the experimental binding free energy by converting the experimental 12,13 while methylation at position 4 seemed an interesting modification to increase the potential interactions of the ligand with binding pocket residues. QUB-00006 was generated based on the structure and position of x0195 in the co-crystal (5R81), and then placed in the receptor structure (Mpro dimer with the PDB code: 71LL); next, the Mpro–QUB-00006 complex was equilibrated using MD simulations . We note that methylation at position 4 of the tetrahydroquinoline core introduces a chiral center, however no significant differences in terms of pocket occupancy between the R) and QUB-00006(S) are −2.73 ± 0.34 kcal mol−1 and −2.72 ± 0.22 kcal mol−1, respectively (pro S2 subpocket and could represent a starting point for structure-based design of novel Mpro inhibitors.The computed absolute binding free energies for QUB-00006(ectively . These rpro binding pocket: (i) the sulfur group of QUB-00006(R) interacts with the oxygen of the carbonyl belonging to the backbone of Glu166 with a distance of 3.3 Å, (ii) the α,α-difluoro moiety points towards His41, and (iii) the sulfur of Met49 establishes a favorable interaction with one of the two fluorines of the substrate (distance 3.3 Å). In fact, the sulfur–oxygen contact observed in our simulations is in agreement with the findings of a study conducted by Iwaoka et al.,68 where they found that a total of 1200 and 626 fragments from the Cambridge Structural Database (CSD) and Protein Data Bank (PDB), respectively, have close intermolecular S–O contacts (with a distance of 3.52 Å or less). Another study analyzing the protein structures deposited in the Protein Data Bank reports 1133 interactions between His and halogen atoms found in 3833 PDB entries with one or more halogenated ligands co-crystallized with a protein.69 Moreover, the strong S–F interaction identified during the simulations is in good agreement with experimentally observed distances for fluorine–sulfur contacts in crystal structures (2.8–3.4 Å).70 It is worth noting that such interactions involving sulfur and halogen atoms are usually better captured with polarizable models than with their classical counterparts.71–73The identified binding mode is defined by several favorable intermolecular interactions occurring between the newly designed ligand and the Min vitro the simulation outcomes.QUB-00006 was then synthesized following the path in The ligand orientation in the MD simulations and the computed hydration ratio of the different atoms of QUB-00006 during ABFE simulations suggest that proton C is solvent exposed, while the protons of the methyl thioether group (group E) and the methyl group at position 4 of the tetrahydroquinoline core (group D) are buried .via WaterLOGSY experiments. In fact, WaterLOGSY epitope mapping confirms that QUB-00006 binds to the protein binding pocket. We leveraged the experimental approach to better identify the region of the ligand in contact with the protein. In pro show a change in the sign suggesting that these protons are in close contact with the protein. Similarly, the aromatic protons A and B undergo a sign inversion. In contrast, the aromatic proton C is not significantly perturbed, which suggests that this position is solvent exposed. The binding mode suggested by NMR is in agreement with the MD-derived hydration ratios confirming the predictive power of our MD-based approach to characterize the binding mode of novel ligands at the experimental level of accuracy  of the more polar QUB-00006-Int-01 compound, we decided to predict its binding conformation at the binding pocket and compute the respective absolute binding free energy. Modification of the molecular scaffolds, especially in fragment-like molecules, might affect the binding mode74 compared to a reference structure (e.g. x0195 as per PDB ID:5R81).Before conducting NMR STD experiments to determine the dissociation constant (R) and QUB-00006-Int-01(S) , which agrees reasonably well with our binding free energy calculations (R) and QUB-00006-Int-01(S) fill up a binding pocket space that is different from the one occupied by QUB-00006. On the other hand, starting with a QUB-00006-like binding mode, we ran an additional absolute binding free energy calculation on an Mpro–QUB-00006-Int-01(R) complex and obtained a binding free energy of −0.9 kcal mol−1. These results suggest that QUB-00006-Int-01 and QUB-00006 might have different dominant binding conformations   with comulations . As showulations . Additioions see  and 5A.pro–ligand interactions  to further explore the conformational space of QUB-00006-Int-01. AS can be used here as an interpretative tool able to gather structural insights on the various potential M−1 of what was previously obtained. The simulations of QUB-00006-Int-01(R) and QUB-00006-Int-01(S) converged to similar ensembles containing several possible binding modes. Clusters 3, 5, and 6 of QUB-00006-Int-01(R) and cluster 4 of QUB-00006-Int-01(S)  is able to capture potential binding orientations of fragment-like compounds in the binding pocket of a protein, and to accurately predict their binding free energies.75 which aims at finding the slow collective variables of the data, and applied it to QUB-00006-Int-01(R). We then used the k-means clustering method on the data projected on this space and built a Hidden Markov State Model (HMSM).76 Three clusters emerged, whose characteristics also show the coexistence of several binding modes of QUB-00006-Int-01(R), one of which corresponds to a distance between C2 and SG below 4 Å. Detailed results can be found in the ESI.‡Then, we analyzed the clustered QUB-00006-Int-01 binding conformations from the adaptive sampling simulations plotted as a function of the distance between the methyl thioether group in QUB-00006-Int-01 and the beta carbon of Gln189, and the distance between C2 (carbon connected to the hydroxyl group) and the sulfur atom (SG) of the catalytic side chain of the Cys145 residue . We notipro.38,63,77 As a matter of fact, a simple chemical modification to QUB-00006-Int-01 would lead to QUB-00006-Int-07 bearing an α,α-difluoro-keto moiety, which is prone to a nucleophilic attack by the vicinal R-SH of Cys145. In order to enable the latter, QUB-00006-Int-07 would need to access the Mpro substrate pocket and adopt a stable binding conformation prior to the covalent binding. Thus, we conducted absolute binding free energy simulations on the Mpro–QUB-00006-Int-07 complex, which confirmed a favorable binding energy of QUB-00006-Int-07 to the Mpro substrate pocket . As reported in Targeting Cys145 with covalent warheads has been used by several researchers to discover novel potent inhibitors of Mpro. Being a fluorogenic assay, compounds with fluorescence quenching properties can suppress the fluorescence signal generated by the protease activity. To eliminate false positive results, we conducted a preliminary counter screen and verified that the tested compound possesses negligible fluorescence quenching effects. Subsequently, to assess the potential inhibitory activity of the compound against SARS-CoV-2 Mpro, increasing concentrations of QUB-00006-Int-07 (0.25–150 μM) were incubated with 20 nM Mpro before the addition of 5 μM FRET substrate. As shown in pro with 50% inhibitory concentration , thus resulting in a fairly potent inhibitor of the Mpro enzymatic activity. The binding of QUB-00006-Int-07 to Mpro was confirmed by electrospray ionization (ESI) mass spectrometry.Our computational findings motivated us to test the compound with a FRET-based proteolytic assay. This assay should detect potent functional binders to the viral Mpro (compound : protein ratio = 10 : 1) was analyzed by ESI-MS under denaturing conditions, and a representative spectrum is provided in pro modified by the presence of the compound (green asterisks) which is therefore covalently linked to the protein given the non-native conditions of the experiment. The nature of the adduct and the molecular mechanism of binding are under investigation and will be the subject of further studies.A preliminary determination of the initial protein showed an experimental mass of 33 796.40 Da, which matches very closely the expected value of 33 796.64 Da calculated from the sequence . The same.g. gem-difluoro, thioether, hydroxyl and methyl groups) to the tetrahydroquinoline scaffold of x0195, and the design and synthesis of novel molecular scaffolds, enabled the exploration of binding pocket boundaries and provided additional information related to druggability of the S2 subpocket. Other molecules were produced over the course of this research but, due to their weaker activity, their detailed analysis is not provided here. Their list can be found in the ESI.Finally, in this work, the introduction of multiple modifications . The results of the innovative scaffold design described here were obtained within three months via a fast-track project that took place in the summer of 2021. It involved a small consortium of theoreticians, organic chemists and drug designers, and demonstrated the effectiveness of a computation-guided synthetic strategy. Indeed, GPU-accelerated high-performance computing platforms can now provide access to high-resolution molecular dynamics simulations, which are able to predict detailed protein conformational maps and provide accurate absolute binding free energy results. Such computations can be further rationalized by means of adaptive sampling simulations, an approach which is able to decipher multiple binding modes. Coupled to NMR, in vitro experiments and machine learning, such high-resolution predictions yield structural insights regarding the design of new active compounds, while offering an atomic level understanding of binding affinities.We presented a computationally driven discovery of a new set of non-covalent and covalent inhibitors of M78,79 of the warhead reaction mechanism38,77,80 leading to the covalent binding of QUB-00006-Int-07, and to optimization of active compounds with the goal of reaching low nanomolar activity.Beyond this preliminary proof of concept study, the next research steps will be devoted to the QM/MM modelingAll data have been provided in the main text and ESI.†L. E. K., Z. J., D. L., T. J. I., D. E. A., B. S., and L. L. performed the simulations. L. L., T. J. I., D. S., and J.-P. P. contributed new code. M. S., M. B., A. S., and M. V. performed NMR and mass spectroscopy experiments. I. K., R. I., A. K., Y. M., A. P., and V. C.-R. performed the synthesis. L. L., B. S., P. R., J. W. P., D. S., and J.-P. P. contributed new methodology. L. L., B. S., F. H., P. R., J. W. P., and J.-P. P. contributed analytical tools. All authors analyzed the data. L. E. K., Z. J., L. L., M. S., A. S., D. S. and J.-P. P. wrote the paper. D. S. and J.-P. P. designed the research.P. R., M. M., L. L., J. W. P. and J.-P. P. are co-founders and shareholders of Qubit Pharmaceuticals.SC-013-D1SC05892D-s001


Dysphania ambrosioides (Silverweed) intoxication in the context of SARS-COV-2 infection in a patient admitted for delirium with Glasgow Coma Score (GCS) of 13/15. This herb was used as an antipyretic to treat COVID-19 persistent fever. The clinical presentation of our patient raised several questions related to the viral or herbal intoxication origin of the confusion syndrome. To our knowledge, this is the first description of toxic encephalopathy after D. ambrosioides ingestionin an adult patient. We report the case of potential  Several neurological manifestations associated with COVID-19 have been reported during this global pandemic. The neuro-invasive potential of the SARS-COV-2 has been described and many case series reported frequent mental confusion especially in intensive care units. Nevertheless, clinicians should not forget to deeply explore the other possible origins of a confusion syndrome. A 39-year-old patient with a history of treated pulmonary tuberculosis followed up initially at home before hospital admission for SARS-COV-2 infection revealed by fever, anosmia, and ageusia. COVID-19 real-time reverse transcriptase-polymerase chain reaction (RT-PCR) test on nasopharyngeal swab was positive. On day 5 of infection, the patient was admitted to the hospital for delirium.The initial clinical examination reported a confused patient with temporospatial disorientation, inconsistent speech, and a GCS of 13/15. His central temperature was at 37.5 °C. The patient’s blood pressure was normal (130/90 mmHg) with a heart rate of 90 bpm and peripheral capillary oxygen saturation of 95%. The clinical examination highlighted a cutaneous maculopapular and erythematous rash affecting the face and the trunk. The neurologic examination was normal.Dysphania ambrosioides - based oral infusions for five days to treat his fever. The brain MRI was unremarkable. Cerebrospinal fluid analysis (CSF) revealed a clear liquid (rock water) with negative direct examination, leukocytes <5 elements /mm3; proteinorachia at 0.45 g/l, and glycorachia at 0.63 g/l. Interestingly, the family interview revealed the use of unspecified amounts of 3 without lymphopenia, ferritin concentration at 200 ng/ml . Coagulation tests, as well as infectious serologies, were negative. Renal and hepatic functions were strictly normal as well as tests for toxins in the urine  which returned negative. Vitamin B1, B6, B9, and B12 dosages were within the normal range. Laboratory tests and hematological parameters were as follows: sodium at 132 mmol/l, C-Reactive Protein at 20 mg/l , neutrophilic leukocytosis at 11.103/mmThe chest CT-Scan showed asymmetric bilateral ground-glass opacities estimated between 5-10% Figure .A probabilistic antibiotic and antiviral treatment (based on Ceftriaxone 6g/day and Acyclovir 10mg/kg/day) was started in the emergency department and stopped upon receipt of the results of CSF analysis. The latter's evolution was marked by the regression of the delirium 24 h after the admission as well as the cutaneous rash. The patient was discharged 48 hours after admission and seen one week later in ambulatory care where his neurological examinations were unremarkable.The clinical presentation of our patient raised several questions related to the viral or herbal intoxication origin of the confusion syndrome. Indeed, in the neurological manifestations associated with COVID-19, the direct damage to the central nervous system by the SARS-COV 2 does not appear to be in the foreground. Nevertheless, the neuro-invasive potential of the virus exists and has been described during this pandemic. Rogers et al. mention frequent mental confusion reaching 65% of patients in intensive care ; their mThe cases of meningoencephalitis directly associated with SARS-CoV-2 are rather rare, ranging from 6% to 9.5% depending on the series . HoweverDysphania ambrosioides is an annual or perennial shrub with a strong aromatic odor cultivated in the subtropical and sub temperate regions around the world [D. ambosioides are used as poultice, for baths, oral ingestion or as tea to treat influenza and other respiratory diseases as well as digestive, urogenital, vascular, and nervous disorders [Dysphania ambroisoides essential oil are α-Terpinene (7-53%), p-cymene (2-10%), ascaridole (16-61%), and ascaridole epoxide. These chemical compounds can vary according to several parameters such as climate and soil. Ascaridole is very poorly soluble in water and can concentrate in the essential oil of D. ambrosoides. Monzote et al. showed that the toxicity of the essential oil from Dysphania ambrosioides is partially related to the inhibition of the respiratory chain complex preferably by caryophyllene oxide while the toxicity of ascaridole is dependent on the availability of redox-active iron. Ascaridole was less toxic to mammalian mitochondria than other major ingredients. However, under the availability of redox-active Fe2+, reduced hemin, and oxidative stress conditions, the toxicity can potentially be increased. [On the other hand, he world -10. In fisorders -14. The creased. -17 D. ambrosioides  is frequently used for its antipyretic, analgesic, hemostatic and antispasmodic effects. However, prolonged and uncontrolled exposure to this plant can lead to more or less significant renal, gastrointestinal, and also encephalic complications depending on the duration of exposure and the dose administered [D. ambrosioides intoxication cases are poorly documented. To our knowledge, only two cases affecting the pediatric population were reported in the literature. Clinically, D. ambrosioides-related toxic encephalopathy may present with mental confusion, behavioral disturbances, vigilance disorders ranging from obtundation to deep coma, myoclonus, and seizures. In all cases, clinical improvement is essential to confirm the diagnosis and given the absence of an antidote, the treatment is purely symptomatic. [In Morocco, nistered . Data ontomatic. -19D. ambrosioides ingestion and symptomatic treatment. All these arguments tend to support the herbal intoxication origin of the confusion syndrome.In our case, the patient presented with a mild form of COVID-19, both brain MRI and CSF were normal, his biological inflammatory parameters were moderate without ionic disorders, with neither hepatic nor kidney function disturbances. His EEG did not show any seizure abnormalities and the evolution of confusion as well as cutaneous rash was rapidly resolutive after cessation of The case of our patient should remind clinicians, especially in the countries where traditional medicine is frequently used, to consider the possibility of medicinal plants intoxication as their prescription, preparation, and consumption are not always obvious nor automatically revealed during anamnesis.


N) inhibitor can inhibit the entire PA endonuclease activity. In this study, we screened the effectivity of PAN inhibitors from the FDA database through in silico methods and in vitro experiments. PAN and mutant PAN-I38T were chosen as virtual screening targets for overcoming drug resistance. Gel-based PA endonuclease analysis determined that the drug lifitegrast can effectively inhibit PAN and PAN-I38T, when the IC50 is 32.82 ± 1.34 μM and 26.81 ± 1.2 μM, respectively. Molecular docking calculation showed that lifitegrast interacted with the residues around PA or PA-I38 T’s active site, occupying the catalytic site pocket. Both PAN/PAN-I38T and lifitegrast can acquire good equilibrium in 100 ns molecular dynamic simulation. Because of these properties, lifitegrast, which can effectively inhibit PA endonuclease activity, was screened through in silico and in vitro research. This new research will be of significance in developing more effective and selective drugs for anti-influenza therapy.Drug repurposing can quickly and effectively identify novel drug repurposing opportunities. The PA endonuclease catalytic site has recently become regarded as an attractive target for the screening of anti-influenza drugs. PA N-terminal (PA N) contains an endonuclease active pocket and plays a crucial role in influenza virus polymerase activity. Drugs that abolish PAN endonuclease activity or disturb the assembly of RdRp can effectively inhibit the replication of the influenza virus [N endonuclease domain is highly conserved among different influenza virus subtypes, indicating that PAN is a promising broad-spectrum anti-influenza therapeutic target because of its ability to inhibit virus proliferation during the initial mRNA synthesis stage [Influenza is an infectious disease that causes 300,000 to 500,000 human deaths globally per year ,2. The Iis stage .N endonuclease inhibitor . Several classes of PAN endonuclease inhibitors researched included: 4-substituted 2,4-dioxobutanoic acids and 3,4-substituted 2,6-diketopiperazines [N endonuclease, was approved by the FDA as an inhibitor of influenza A and B after proving effective in clinical trials [The three classes of FDA-approved anti-influenza drugs are: neuraminidase inhibitor (oseltamivir and zanamivir), M2-ion channel inhibitor (adamantanes), and PAerazines , flutamierazines ,9,10, N-erazines , catechierazines , etc. Bal trials .N endonuclease and Baloxavir acid were disturbed after the Ile 38 mutated to Thr [N endonuclease domain is the primary mutant that leads to influenza’s resistance to Baloxavir acid. This resistance reduces the effectiveness of Baloxavir acid as an anti-influenza drug [A mutation in the influenza viral genome led to the influenza’s subsequent drug resistance. The hydrophobic interaction residues between PAd to Thr . The I38nza drug . Using tDrug repurposing research can effectively identify new drug repurposing opportunities, quickly expand the drug market, and reveal new commercially valuable uses for existing drugs . Some exN endonuclease inhibitors from the FDA-approved database through in silico methods and in vitro experiments. PAN inhibitors can inhibit the entire PAN endonuclease activity. Since the purpose of this study is to identify drugs that overcome resistance, we screened inhibitors that target both PAN and mutant PAN -I38T. Experimental tests have verified that the drugs lifitegrast and saquinavir have an inhibitory effect on PAN, while the drugs lifitegrast and conivaptan have an inhibitory effect on PAN-I38T. In addition, molecular docking shows the interaction mechanism between lifitegrast and the active site of PAN or PAN-I38T. Research suggests that lifitegrast may be a potential anti-flu drug. Finally, the method employed in this work could be utilized as a fast and viable strategy for accelerating research in the treatment of influenza.This research screened the effectivity of PAN endonuclease inhibitor structure, we performed the virtual screening protocol based on the PAN and mutant PAN-I38T structure. The residue Ile38 was not essential in either metal-ion binding or in catalytic activity. The I38 substitution does not block the endonuclease reaction but offers resistance to the inhibitor because the I38 side chain interacts with the Baloxavir acid. After the virtual screening, the compounds interacting with PAN or PAN-I38T were sorted by affinity score. The top-ranked compounds listed in N and PAN-I38T as hit compounds. Finally, six compounds, saquinavir, conivaptan, lifitegrast, rifaximin, dutasteride, and lurasidone, were used for further basic gel endonuclease determination. In comparison, three compounds, Teniposide [To develop the new PAniposide , Simepreniposide , and Nilniposide , were fiN or PAN-I38T, respectively. Next, we incubated different concentrations of compounds with PAN or PAN-I38T and then detected the cleavage of a substrate . Resistance develops when the hydrophobic interaction residues between PAN endonuclease and Baloxavir acid are disturbed after the Ile 38 mutates to Thr. Therefore, we used the mutant type of PAN-I38T as the virtual screening target to avoid drug resistance. We used the FDA database to effectively identify drug repurposing opportunities. Then, we discovered a potent inhibitor, lifitegrast, which differs from Baloxavir acid in that it has the ability to interact equally with both PAN and PAN-I38T.Researchers have persistently searched for a drug that could be used to treat all types of influenza viruses, including influenza A, influenza B, and influenza C, along with their mutants. In this study, we used PAN and PAN-I38T. Molecular simulation displayed that lifitegrast interacted with the active site of PAN or PAN-I38T. Further molecular dynamic simulation analysis suggested that the bindings between PAN/PAN-I38T and lifitegrast were stable.Gel-based endonuclease inhibitory assay showed that lifitegrast effectively inhibits the substrate cleavage of both PAHowever, there are still numerous challenges. Subsequent viral experiments in vivo need to be implemented in order to verify the reliability of experiments in vitro. Meanwhile, lifitegrast has been used for the treatment of dry eye, but further toxic experimentation is required before lifitegrast can be used as a treatment for the influenza virus. N endonuclease inhibitor from the FDA database through in silico and in vitro experiments. The findings suggest that lifitegrast may be a potential anti-flu treatment drug because it could solve the drug-resistant properties, which are superior to currently available flu treatments, such as Baloxavir acid. Finally, the method adopted in this work could be utilized as a fast and viable strategy for accelerating research on the treatment of influenza.In this research, we screened the effectivity of the PAN endonuclease protein crystal structure (PDB ID: 6FS6) and I38T mutant (PDB ID: 6FS7) in the PAN subunit were obtained from the RCSB Protein Data Bank database [The PAdatabase . One thodatabase . Virtualdatabase , with thCompounds saquinavir, conivaptan, lifitegrast, rifaximin, dutasteride, and lurasidone were purchased from Aladdin  or Yuanye Biotech . Substrate DNA was purchased from Takara Bio lic. N endonuclease domain from influenza A type virus (H1N1)  was cloned into the pET-28a vector, expressed in E. coli BL21 (DE3), and purified by IMAC. The positive clone was used for large-scale expression. Cells were expressed in LB medium at 37 °C for 4 h with 1 mM IPTG. Before the endonuclease activity assay, the protein sample was concentrated, and the buffer changed  by ultrafiltration. The protein concentration was measured by the BCA method. The PAN-I38T was cloned from the PAN through site-directed mutagenesis. The mutant PAN-I38T was expressed and purified the same as the wild PAN.The PAN endonuclease activity was detected through the digestion of a single-strand DNA substrate. In the digestion reaction, PAN or PAN-I38T was mixed with ssDNA in a digestion buffer that included 2 mM MnCl2. To determine the concentrate of PAN or PAN-I38T for endonuclease activity assay, 0–1.5 μM protein and 100 ng ssDNA were incubated at 37 °C for 60 min. After digestion, the samples were fractioned by 0.8% agarose gel and visible by a stain. Image J was used to determine the density of substrate ssDNA. Finally, 1.5 μM PAN or PAN-I38T were digested at 100 ng ssDNA in 10 μL volume for 60 min.The PAN or PAN-I38T and 100 ng ssDNA mixed with different concentrations of compounds were used for the experiment test. Samples without inhibitors were used as the no-compound control. Samples without protein were used as the substrate control. After digestion, the samples were loaded on the agarose gel for separation and were visible by a stain. The amount of remaining ssDNA was determined by image J. The IC50 value was calculated by GraphPad Prism 6.0 through non-Linear regression.Compounds screened from the FDA library analysis determined the inhibition of endonuclease activity. In total, 1.5 μM PAN and PAN-I38T with or without compounds was performed using GROMACS version 2018 with the CHARMM36 all-atom force field (28 March 2019) to investigate the inhibition mechanism [The MD simulation of PAechanism ,30. The echanism . Two addechanism , RMSD isechanism ,34,35.50 was calculated by GraphPad Prism 6.0 using non-linear regression. The data were represented as mean ± SD.The IC


Nitric oxide (NO) has been shown to stimulate differentiation and increase the survival of ganglionic sympathetic neurons. The proportion of neuronal NOS-immunoreactive sympathetic preganglionic neurons is particularly high in newborn rats and decreases with maturation. However, the role of NO in the development of vascular sympathetic innervation has never been studied before. We tested the hypothesis that intrauterine NO deficiency weakened the development of vascular sympathetic innervation and thereby changed the contractility of peripheral arteries and blood pressure level in two-week-old offspring. Pregnant rats consumed NOS inhibitor L-NAME (250 mg/L in drinking water) from gestational day 10 until delivery. Pups in the L-NAME group had a reduced body weight and blood level of NO metabolites at 1–2 postnatal days. Saphenous arteries from two-week-old L-NAME offspring demonstrated a lower density of sympathetic innervation, a smaller inner diameter, reduced maximal active force and decreased α-actin/β-actin mRNA expression ratio compared to the controls. Importantly, pups in the L-NAME group exhibited decreased blood pressure levels before, but not after, ganglionic blockade with chlorisondamine. In conclusion, intrauterine L-NAME exposure is followed by the impaired development of the sympathetic nervous system in early postnatal life, which is accompanied by the structural and functional remodeling of arterial blood vessels. Nitric oxide (NO) is an important regulatory molecule in mammalian organisms. Physiologically relevant NO is generated by two constitutive nitric oxide synthase isoforms: neuronal (nNOS) and endothelial (eNOS) ,2. nNOS In early postnatal ontogenesis, the regulation of the circulatory system has many unique characteristics compared to adult organisms, one of which is the augmented role of NO in vasomotor control ,4. Such Data in the literature suggest the role of NO in the control of sympathetic innervation development. Cell culture studies have demonstrated that NO can stimulate differentiation/neurite growth and increase the survival of nerve cell precursors and sympathetic neurons ,16,17,18ω-nitro-L-arginine methyl ester (L-NAME) to pregnant females [In the present study, we tested the hypothesis that NO deficiency in the developing organism weakens the formation of vascular sympathetic innervation, which may result in the decreased contractility of peripheral arteries and the lowering of blood pressure levels. Intrauterine NO deficiency was achieved through the chronic supplementation of N females ,26,27. L females . Experim females ,10,29.The body weights of females in the control group and L-NAME group were almost similar at the seventh gestation day GD7, a. ConsumSystolic blood pressure and heart rate were not different between the control and L-NAME groups before L-NAME treatment c,d, GD7.Notably, the consumption of L-NAME led to a decrease in the content of NO metabolites in the blood serum of females on GD18-19. Any notable changes in several metabolic parameters, such as triglycerides and total cholesterol, were not observed after L-NAME consumption .p > 0.05). Offspring born from L-NAME-treated females (“L-NAME offspring” in the following description) exhibited a reduced body weight at postnatal day 2 (PND2) but reached control values by PND14 . They are referred to as two-week-old pups in the following text.To identify whether NO deficiency affects the development of sympathetic innervation, we evaluated the density of adrenergic fiber plexus in isolated saphenous arteries in two-week-old pups. This resistance-type small artery predominantly provides blood flow to the skin of the foot and presents dense sympathetic innervation in adult rats ,10.Sympathetic innervation density was estimated by the total length of the skeletonized nerve fibers . Figure 100, see n = 17) vs. 296 ± 8 μm (n = 18) .As the next step of our study, the functional characteristics of two-week-old offspring saphenous artery were examined. The inner diameters of relaxed saphenous arteries . Chlorisondamine caused a drop in mean arterial pressure in both groups, and the mean arterial pressure was no longer different between groups a. NotablWe showed that the body weight gain in dams was not affected by the administration of L-NAME from GD10 until a delivery. Notably, L-NAME treatment was able to cause a rise in systolic blood pressure in dams, even though the dose used in this experiment was relatively lower than that reported in the literature ,35,36,37Vasoconstriction and limited uteroplacental perfusion may lead to intrauterine growth restriction of the fetus ,40. Our The relevance of our NO deficiency model was also confirmed by a decrease in the blood content of nitric oxide metabolites in L-NAME-treated females. The concentration of nitric oxide metabolites in blood sera of the offspring born from L-NAME-treated rats was also significantly reduced compared to controls soon after birth (PND2), which can be explained by the ability of L-NAME to cross the placenta, in addition to maternal NO deficiency . PreviouIn addition to its important vasomotor role, NO was shown to promote differentiation and increase the survival of nerve cell precursors and sympathetic neurons ,17,18,44To assess whether NO deficiency would influence the development of sympathetic innervation, the density of sympathetic nerve fibers was evaluated in the isolated saphenous artery. Notably, in adult rats, the saphenous artery exhibits dense sympathetic innervation and develops strong contractile responses to the stimulation of sympathetic nerves ,45. AccoOur data demonstrate that the total length of adrenergic fibers in the saphenous artery of two-week-old pups born from L-NAME-treated females is reduced compared to controls. Impaired vascular innervation development can be a long-lasting effect of L-NAME exposure, which is preserved after the disappearance of the substance from pup’s tissues. Thus, NO may control the development of vascular sympathetic innervation during early postnatal ontogenesis. Notably, the sympathetic nerve plexus in the peripheral arteries of two-week-old rats is still developing ,10,29. TIt is well known that the trophic action of sympathetic nerves promotes the growth and differentiation of vascular smooth muscle cells and vessel remodeling ,46. Our We have shown that saphenous arteries from two-week-old L-NAME offspring have a smaller inner diameter and maximum contractility, the latter often correlating with reduced media thickness . The assTrophic action of the sympathetic nerves was shown to increase the expression of smooth-muscle-specific α-actin and myosin ,13, prom1-adrenoceptor agonist methoxamine in L-NAME offspring was considerably higher than that in the control group. We considered that such arterial hypersensitivity was due to the altered response of smooth muscle, not by the attenuated anticontractile effect of the endothelium. In a one- or two-week-old rat saphenous artery, the endothelium weakens the effects of vasoconstrictors due to the release of NO [p < 0.05, unpaired Student’s t-test). This points to the higher sensitivity of smooth muscle cells to methoxamine in L-NAME offspring compared to controls.The arterial sensitivity to αse of NO . In our 1A-adrenoceptors [1A-adrenoceptor mRNA in arterial tissue was not changed by maternal L-NAME treatment. The mechanism of denervated arteries hypersensitivity downstream of the receptors may include membrane depolarization, and, consequently, the augmented activation of voltage-gated calcium channels [The arteries of L-NAME offspring are more sensitive to methoxamine because of their sparse sympathetic innervation. Indeed, the augmented sensitivity of chronically denervated arteries to adrenoceptor agonists was shown in several studies ,48,49,50oceptors ,52, whicoceptors . Presumachannels . FurtherThus, intrauterine L-NAME exposure reduces the density of sympathetic innervation of the saphenous artery. This may diminish the trophic influence of sympathetic nerves, and, consequently, cause a delay in arterial maturation, manifested in decreases in the inner diameter, maximal active force, and α-actin/β-actin expression ratio.Sympathetic nervous system activity contributes significantly to maintaining arterial pressure level. In rats, the sympathetic control of blood pressure levels has been observed from the ages of 1 to 2 weeks ,8.To identify whether the reduced density of sympathetic innervation observed in the saphenous artery of L-NAME offspring would be manifested at the systemic level, we performed the measurements of systemic hemodynamic parameters. We have shown, for the first time, that the mean arterial pressure levels in two-week-old L-NAME offspring are significantly lower in comparison to a control group. We have also demonstrated that differences in mean arterial pressure between the two experimental groups disappeared after ganglionic blockade by chlorisondamine.The heart rate values in the two groups were similar both before and after the ganglionic blockade; therefore, the depressor effect of chlorisondamine primarily reflected the sympathetic influence on peripheral vascular tone. Therefore, the difference in arterial pressure levels between two groups of pups before, but not after, ganglionic blockade allows us to conclude that reduced blood pressure levels in L-NAME pups are due to a smaller neurogenic component of vascular tone.1-adrenoceptor agonist were not reduced in two-week-old L-NAME pups; therefore, we suggest that smaller sympathetic components of vascular tone in L-NAME pups were due to the inhibition of sympathetic ganglionic neuron function because of their relative immaturity.Neurogenic components of blood pressure level are determined by the functions of sympathetic neurons (their discharge and neurovascular transmission) and vascular reactivity to the influence of neurotransmitters. Arterial responses to intermediate (“regulatory”) concentrations of the αHowever, heart rate responses to the ganglionic blockade were not changed in L-NAME pups compared to control pups. Notably, the heart rate in rat pups is greatly influenced by adrenal catecholamines . If adreThus, intrauterine L-NAME exposure weakens the sympathetic control of peripheral vasculature in two-week-old rats. Importantly, our results suggest a weak development of sympathetic innervation in two-week-old L-NAME offspring to be typical not only for the saphenous artery, but also for other vascular regions.Experiments were performed using Wistar rats in accordance with the European Convention on the protection of animals used for scientific purposes (EU Directive 2010/63/EU). All animal procedures were approved by the Moscow State University committee on animal welfare . Sexually mature (3–4-month-old) male and female Wistar rats were obtained from the vivarium of the Research Institute of General Pathology and Pathophysiology, Russian Academy of Sciences , and then bred in the laboratory animal unit of the Biological Faculty, Moscow State University. The animals were maintained with a 12/12 h light/dark cycle and had free access to normal rodent chow .Intrauterine NO deficiency was simulated by the suppression of NO production in the female body during the second half of pregnancy with the nitric oxide synthase inhibitor L-NAME . For mating, three females and two males were housed together overnight. The onset of pregnancy was determined by the presence of sperm in the vaginal smear the next morning (this day was considered GD1). On GD10, females were randomly assigned to control or L-NAME groups. Females in the L-NAME group received L-NAME in drinking water (at a concentration of 250 mg/L) from GD10 until delivery; females in the control group received tap water. During the pregnancy, the females of both groups were regularly weighed, and their water intake was recorded.Female systolic BP and heart rate were also recorded using tail-cuff plethysmography . The first pre-pregnancy recording served to familiarize the animals with the procedure. During the pregnancy, the recordings were performed on GD7, GD15, and GD20. At every time point, the measurements were performed at least 5 times for each rat, and the obtained values were averaged. On GD18–GD19, blood samples (about 300 µL) were taken from the incision of females’ tail tips to determine biochemical parameters and the content of NO metabolites.On the day of delivery (considered as PND1), the number of pups in each litter was limited to eight. Trunk blood was collected from one pup per litter (both sexes) for further analysis at PND2. The litters were regularly weighed from PND2 to PND14. Male pups aged 14 to 16 days old were further used in experiments on isolated arteries and blood pressure recordings.g for 15 min. Serum was collected and stored at −20 °C until further analysis.To obtain serum, blood samples were kept for 20 min at room temperature, and for an additional 40 min at +4 °C, and then centrifuged at 1350× 3− to NO2− using VCl3 simultaneously with the Griess reaction [The concentrations of total cholesterol and triglycerides were measured by enzymatic colorimetric methods using reagents from Hospitex Diagnostics . The determination of NO metabolite contents was carried out using the Griess method after the deproteinization of the samples, extraction of lipids, and the reduction of NOreaction . OpticalTo visualize sympathetic nerves in the wall of the saphenous artery, we used a method based on the formation of a fluorescent complex resulting from the interaction of catecholamines with glyoxylic acid ,56,57.The saphenous arteries were isolated, carefully cleaned from the surrounding tissue, and then incubated in 0.1 M phosphate-buffered saline solution (pH 7.4) supplemented with glyoxylic acid (2%) and sucrose (10%) for 30 min at room temperature. Then, the arteries were flattened on a slide with the adventitia upward, dried (30 min in a jet of warm air and 5 min at 100 °C), and overlaid with mineral oil and a cover glass.A confocal microscope Zeiss LSM700 with air Plan-Apochromat 20×/0.8 M27 objective  was used to visualize glyoxylic-acid-induced fluorescence in the immersed preparations. The emitted fluorescence was detected in confocal mode with a 0.56 μm pinhole in 405–480 nm (maximum at 435 nm) wavelength range and was induced by diode excitation with a 405 nm laser. Confocal 2048 × 2048 pixel images including 25 stacks that covered the entire thickness of tissue samples were recorded using Carl Zeiss ZEN 7.0 software.Collected data were analyzed offline using ImageJ 1.50i software. Open-source Bio-Formats Explorer ImageJ plugins (imagej.net/Bio-Formats) were used to handle images. After background subtraction and binarization, skeletonization and skeleton analysis (imagej.net/Skeletonize3D) were performed in order to estimate the total length of catecholamine-positive fibers in the saphenous artery wall.2, 0.1 mM; MgSO4, 1.0 mM; NaH2PO4, 1.2 mM; EDTA, 0.025 mM and HEPES, 5.0 mM (pH = 7.4). The myograph chambers were heated to 37 °C and continuously bubbled with 5% CO2 in O2 to maintain pH at a constant level of 7.4. Data were recorded at 10 Hz using an analogue-to-digital converter  and PowerGraph 3.3 software .Saphenous arteries were carefully cleaned from the surrounding tissue, cut into 2 mm long segments, and mounted in a wire myograph  for isometric force recording. The procedures of isolation and mounting were performed in a preparation solution containing: NaCl, 145 mM; KCl, 4.5 mM; CaCl3, 4.5 mM KCl, 1 mM MgSO4, 1.2 mM NaH2PO4, 5.5 mM D-glucose, 0.1 mM EGTA and 5 mM HEPES) in the presence of 1 μM NO donor DEA/NO. Each arterial segment was stretched to 0.9d100 , corresponding to the development of the maximum active force [The standard start-up of each experiment included normalization procedures  and activation. To achieve the full relaxation of arterial smooth muscle, normalization was performed in a calcium-free solution  noradrenaline ; (ii) methoxamine (100 μM) for 5 min followed by acetylcholine (from 10 nM to 10 μM), to confirm the endothelium functionality; and (iii) methoxamine (10 μM). Noradrenaline, methoxamine and acetylcholine were obtained from Sigma .During activation and subsequent experimentation, arterial segments were kept in a solution containing: NaCl, 120 mM; NaHCOω-Nitro-L-arginine, Alexis Biochemicals, San Diego, CA, USA). Subsequently, a second determination of the concentration-dependent response to methoxamine was performed at the same concentration range.Twenty minutes after the completion of the activation procedure, the concentration-dependent response to methoxamine was determined at the range from 0.01 to 100 μM of the drug. In some experiments, it was followed by a washing step and additional incubation with or without 50 μL of 100 μM L-NNA (Nmax (maximum response). To estimate the effect of L-NNA on contractile responses, all active force values obtained during the second concentration-dependent response were expressed as the percentage of the maximum active force in the first concentration-dependent response.To calculate the active force values, the force value at the fully relaxed state was subtracted from all the measured values. Then, individual concentration–response relationships were fitted to a sigmoidal dose–response (variable slope) equation using GraphPad Prism 7.0  to obtain pD2 (the negative logarithm of EC50) and ETM solution  at −20 °C until the next process. Each sample included two arteries obtained from a two-week-old pup. RNA was extracted using the ExtractRNA Kit , according to the manufacturer’s instructions, and processed with DNase I . RNA concentrations were measured with a NanoDrop 1000 ; then, all samples were adjusted to a concentration of 40 ng/μL. Reverse transcription was performed using the MMLV RT Kit  according to the manufacturer’s instruction. qPCR was performed in the RotorGene6000 using qPCRmix-HS SYBR . Primers used in this study were synthesized by Evrogen and are listed in Saphenous arteries were quickly isolated and placed in RNAlater−Ct, where E is the primer efficiency and Ct is the cycle number at which the product fluorescence rose above the threshold level. These values were normalized to the expression value for housekeeping gene Rplp0, detected in the same sample. All values were expressed as the percentage of the mean value in the control group.mRNA expression levels were calculated as EHemodynamic measurements were performed in urethane-anesthetized two-week-old pups . Successful anesthesia throughout the experiment was indicated as the absence of pupil and flexion reflexes with a potentially painful effect on the foot.For arterial BP measurements, a polyethylene catheter (PE5-PE50) was inserted into the right carotid artery. To avoid clotting, the arterial catheter was continuously flushed with heparinized saline (50 U/mL) using a syringe pump ; the infusion rate was 0.08 mL/h. For drug injections, a catheter (PE5-PE50) was inserted into the right jugular vein. Body temperature was maintained at 36–37 °C with a heating blanket throughout the experiment.Arterial BP was continuously recorded at 1000 Hz using a BLPR2 transducer  and a USB-6211 analog-to-digital converter . Data recording and processing were performed using custom-made software written in LabVIEW 2011 .During the initial 15–20 min, arterial pressure and heart rate were allowed to stabilize. Then, the ganglionic blocker chlorisondamine  was injected through a venous catheter, and a 15 min recording was obtained. The blood pressure signal was processed to estimate the mean arterial pressure and heart rate in each cardiac cycle. Hemodynamic parameters were averaged in time intervals taken before chlorisondamine administration  and 10 min after chlorisondamine administration (2 min).t-test or two-way ANOVA. Differences were considered statistically significant at p < 0.05; n is the number of rats in the group.Statistics were calculated using GraphPad Prism 7.0 software . The normality of data distribution was confirmed using the Shapiro–Wilk test. Data are presented as the mean ± S.E.M. The differences between the two groups were assessed using the Student’s In conclusion, our novel findings demonstrate that NO deficiency in early ontogeny reduces the density of the adrenergic nerve plexus in peripheral arteries. This weakens sympathetic control of vascular tone and reduces the trophic influence of sympathetic nerves, which in turn causes structural and functional remodeling of the peripheral vasculature.It should be mentioned that NO synthase inhibition is an established animal model of preeclampsia ,26. In o


Lycium barbarum berries have been utilized in Asia for many years. However, the mechanisms of its lung-defensive properties are indeterminate.L. barbarum polysaccharide (LBP) could weaken Pseudomonas aeruginosa infection-induced lung injury.We investigate whether and real-time quantitative polymerase chain reaction, Real-time qPCR (Q-PCR). Flow cytometry was used to test cell apoptosis. Moreover, Balb/c mice were used to evaluate the tissue injury. We used hematoxylin-eosin staining and immunofluorescence to detect the expression of related proteins and tissue damage in mouse lungs and spleen.Mice primary air-liquid interface epithelial cultures were pretreated with LBP and subsequently treated with pyocyanin (PCN). Lung injury, including apoptosis, inflammation, and oxidative stress, was estimated by western blot, enzyme-linked immunosorbent assay, TNF), IL-6, and IL-8. Our study confirms the ability of LBP to retard PCN-induced injury in mice lung and spleen.The flow cytometric analysis shows the potential of LBP to reduce time-dependent cell death by PCN. Mechanistically, LBP reduces PCN-induced expression of proapoptotic proteins and caspase3 and induces the activation of Bcl-2 in mice bronchial epithelial cells. Similarly, LBP reduces PCN-induced intracellular reactive oxygen species (ROS) production. Moreover, LBP inhibits the production of inflammatory cytokines, Interleukin (IL-1β), Tumor Necrosis Factor (The inhibition of PCN-induced lung injury by LBP is capable of protecting mice cells from injury. L. barbarum polysaccharide on pyocyanin infection.Investigating the effects of Using air-liquid culture as an infection model.Indicating the potential applicability of LBP as an antimicrobial agent.Lycium barbarum (goqi or wolfberry), a member of the eggplant family, is a fruit that has long been used as tea and herb in China and as a dietary supplement for functional food ingredients or micronutrients (L. barbarum polysaccharide (LBP) is the main active ingredient of L. barbarum, which has many biological activities, such as antioxidant and hypoglycemic effects and anticancer activity , which bind to the cell’s receptors surface by receptor-binding regions , which reduces apoptosis . Furtherry tract . At the ry tract , 13. In P. aeruginosa infection. We aim to investigate the potential of LBP pretreatment in alleviating P. aeruginosa. Animal experiments were used to elicit the role of LBP in antagonizing P. aeruginosa infection. Our study scientifically proved that LBP is the main driver of the immunity function of Lycium barbarum.In this study, we generated an air-liquid interface (ALI) culture system of mouse bronchial epithelial cells to analyze their morphological composition. We used the ALI culture system to explore further the immunological response and host cells’ damage after LBP of 95% purity was purchased from Shanghai Biological Co. The LBP was dissolved in ultrapure water and stored at 4°C. All analytical reagents were used directly without further purification. Fresh stock solutions were prepared after 15 days.FBS culture, and the cells on the tissue culture plate incubate the fibroblasts in 5% CO2 for 2–4 h at 37°C. A 500 g of non-disciple cells was centrifuged at 4°C for 10 min, and the cells in the bronchial epithelial cell growth medium were collected. We used an automated vision-based cell counter to determine the total number of cells. P. aeruginosa was used to infect the 6-week ALI cultures. Cells were infected on the culture model’s apical surface for 20 min at a 30-fold multiplicity infection rate. Culture medium containing uncultured P. aeruginosa was removed from the top of the culture. P. aeruginosa was cocultured with epithelial cells for 36 h, and the cells were collected after coculture. The morphology of the cells and bacterial colonization were observed by hematoxylin-eosin staining. All staining reagents were obtained from Sigma-Aldrich . We observed mice bronchial epithelial cells’ characteristics by utilizing immunofluorescent staining of tubulin4 and keratin14. PAS staining was used to detect glycogen secretion in cells. Tubulin 4, keratin 14, and PAS staining: Periodic Acid-Schiff stain (PAS) staining were assessed in mice bronchial epithelial cells.All animal experimental procedures were performed at Ningxia University. This study was approved by the animal ethics committee of Ningxia University with the approval number 2018-019. Sixty (60) Balb/c mice  were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences. The animals were housed in a controlled temperature environment. We developed the mice airway epithelia’s primary culture at the ALI , 13. TenP. aeruginosa  were added to the cells and incubated overnight at 4°C. After removing the primary antibody, the cells were washed three times using PBS. Alexa Fluor 594-labeled antibody (green) and Alexa Fluor 594-labeled antibody  were added to the cells as fluorescent secondary antibodies to detect primary antibodies. The fluorescent secondary antibodies were removed and washed three times with PBS, and the membrane was fixed on a glass slide by Vectashield Mounting Medium  with 4’ 6-diamidino-2-phenylindole (DAPI). Colocalization images were acquired by the Leica TCS SP2 A0BS confocal system and analyzed by the Leica Confocal Software v. 2.6.1 .The cells were fixed with 4% paraformaldehyde for 15 min at room temperature. The fixed cells were washed with PBS three times and then permeabilized with 0.3% Triton X-100 for 20 min. Furthermore, the cells were blocked in 5% ordinary goat serum for 1–2 h at room temperature. Rabbit antikeratin 14 and PAS antibodies , and rabbit anti-3 cells/well. ALI-cultured bronchial epithelial cells were incubated with 5% CO2 at 37°C for 2 h. The PCN-treated group was subsequently treated with a gradient concentration of PCN at the time points 6, 12, and 24 h. The LBP-pretreated group was pretreated with LBP at gradient concentration for 2 h before adding PCN. Cell activity was detected by the 3--dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) method according to the kit instructions. Briefly, 20 μL MTT  was added to the previous section’s processing groups. The MTT-added medium and the cells were incubated at 37°C for 4 h. The plate was read at 570 nm using a microplate reader , according to the reference wavelength. Untreated cells were used as controls, and the wells with PBS served as blank control wells. The average optical density (OD) was subtracted from the OD of the sample compared with the control cells’ OD. We used the following formula to calculate the percentage of cell viability: average density (OD) of the treated group − blank/mean OD of control cells × 100%.The ALI-cultured bronchial epithelial cells were plated in 96-well plates at a concentration of 5 × 10BCA) Protein Assay Kit was employed to determine the content of total protein in lysis cellular extracts (http://rsbweb.nih.gov/ij/). Antibodies used for blotting were Bcl-xl, 1:1,000; Bcl-2, 1:500; caspase3, 1:1,000; caspase9, 1:500 ; cytochrome C, 1:1,000 ; and PARP-1, 1:500 .Cells were lysed in a cell lysis buffer to obtain the total cellular extracts. The Pierce bicinchoninic acid  amplification was performed on the ABI 7500 Fast Thermal Cycler , according to the SYBR Green PCR Kit . PCR cycle was conducted for 30 s at 95°C, followed by 40 cycles at 95°C for 5 s and an annealing/extension step at 60°C for 15 s. The primer was designed by the Shanghai Sango Company . The specific primers are shown in The total RNA from mouse bronchial epithelial cells was isolated using TRIzol3 cells/well. PCN (50 μM) was added to the bronchial epithelial cells and incubated with PCN at 5% CO2 atmosphere at 37°C for 24 h after LBP treatment for 2 h. Twenty-five micromolar DCFH-DA was added to the plate, and the treated cells were incubated with DCFH-DA at 37°C for 30 min. The fluorescence was measured using a Microplate Reader . According to the kit instructions, excitation and emission were set to 485 and 535 nm, respectively. Immunofluorescence microscopy was used to capture the reactive oxygen species (ROS) reaction. The average level of ROS was measured using a ROS assay kit DCFH-DA . Approximately 3 × 105 cells/well were seeded in 6-well plates overnight, followed by PCN treatment with or without LBP pretreatment for 24 h. DCFH-DA was diluted to a final concentration of 10 μM. The cells were collected and suspended in diluted DCFH-DA in the dark at 37°C for 30 min and washed three times with PBS. The resulting samples were analyzed using an Accuri C6 Flow Cytometer .The ALI-cultured bronchial epithelial cells were plated in 96-well plates at a concentration of 5 × 102 atmosphere at 37°C for 24 h. The above-mentioned PCN concentration (50 μM) was added to the cell cultures. The cells were incubated in a 5% CO2 in the atmosphere at 37°C for 24 h after LBP treatment for 2 h. Subsequently, the culture supernatant for inflammatory factors detection was collected. According to the kit instructions, TNF, IL-6, and IL-8 concentrations in the cell supernatant were also determined by enzyme-linked immunosorbent assay (ELISA).PCN (50 μM) was added to the mouse bronchial epithelial cells medium and incubated in a 5% COn = 9). Group I animals were set as the blank control group and treated with 50 μL of physiological saline. Group II animals were set as the PCN treatment group and treated with 50 μg/50 μL PCN. Group III animals were set as the LBP treatment group and treated with LBP at a dosage of 3 mg/kg. Group IV animals were set as the P. aeruginosa treatment group and treated with plaque forming unit (PFU) of 1 × 106 colony forming unit (CFU). Group V animals were set as the PCN treatment group after LBP pretreatment, and the treatment conditions were as described above. The Group VI animals were set as the P. aeruginosa treatment group after LBP pretreatment, and the treatment conditions were as described above. All the above treatments were imposed on the mice by intranasal route for 7 days. According to the aforementioned groups, for 7 days, the mice were sacrificed by cervical dislocation after treatment imposition. The mice were dissected after simple disinfection, and the lung and spleen tissues were collected for further morphological analysis. The lung and spleen sections were blocked at room temperature using saline containing 0.1% bovine serum albumin (BSA). The sections were gently incubated for 2 h using the following primary antibodies: Bcl-2 (Abcam), Caspase-3 , and Cyt-c . Furthermore, we removed the primary antibodies, thoroughly washed the membranes with 0.5% TBS-Tween 20, and subsequently incubated and shook them gently for 2 h after adding a biotinylated secondary antibody. At least five slices per organism were manufactured for each treatment group. We captured images at 200× or 400× using the Zeiss microscope and ZEN software.The animals were randomly divided into six groups .All data collected were obtained from at least three independent experiments for each condition. The GraphPad Prism version 7.0  was used for data analysis and graphic images. Statistical evaluation of the data was performed using a in vitro bronchial epithelial cells in mice, we identified the epithelial cell type by keratins4 and tubing4. PAS staining was used to detect glycogen secretion. The expression profile of the site depends on the differentiation level. Similar to the natural mouse bronchial tree, mouse bronchial epithelial cultures showed keratin 14 expression only in the PAS (characteristic features of the remnant cells) of the basal layer  after a 6 h infection . The expression of Bcl-2 and Bcl-xl decreased in PCN-infected cells (P < 0.05) increased in the LBP pretreated group. LBP could also decrease the expression of pro-apoptosis cytokines. The expression of the cytokines Caspase3, p62, Beclin-1, PARP, BAD, mTOR, TGF-β, and NF-κB was decreased in cells pretreated with LBP compared to the LBP untreated PCN infection group (P < 0.001).Caspase3, p62, Beclin-1, PARP, BAD, mTOR, TGF-β, NF-κB, Bcl-2, and Bcl-xl are cytokines that are important for apoptosis control. The expression of the cytokines Caspase3, p62, Beclin-1, PARP, BAD, mTOR, TGF-β, and NF-κB was induced in cells infected with PCN infection compared to the uninfected control group (ed cells  comparedP < 0.05) suppressed the expression of active caspase-3 . The proportion of apoptotic cells was 76.11% in cells pretreated with LBP and exposed to PCN infection for 24 h at a concentration of 50 μM (The percentage of apoptotic cells (annexin – V + PI + and annexin – V + PI -) was exposed to PCN treatment at a concentration of 50 μM at 84% and 57% on the treated cells for 24 h . Epithelof 50 μM . These dP < 0.05, P < 0.01).For the characterization of cytokine levels in PCN-induced cellular damage and LBP-mediated protection, IL-1β, TNF, IL-6, and IL-8 secreted protein levels were measured by ELISA in all groups at 24 h. PCN increases the secretion of IL-1β, TNF, IL-6, and IL-8 after 24 h treatment . ParticuP. aeruginosa inhibit Bcl-2, Cytc, and Caspase-3, we used immunofluorescence of lung and spleen tissues to examine the interaction between LBP, PCN, and P. aeruginosa. The results indicate that the expression of Caspase-3 and cytochrome c increased under PCN and P. aeruginosa infection but decreased when pretreated with LBP.To elucidate the mechanism by which PCN and P. aeruginosa and LBP protection, we endotracheally induced Balb/c mice with LBP, PCN, and P. aeruginosa. The lung and spleen tissues in mice were analyzed by staining with hematoxylin-eosin. Normal pulmonary tissues were observed in the mice in the control group  has been used in traditional Chinese medicine for a long time. LBPs are important active constituents of L. barbarum. However, there are limited reports concerning the effects of LBPs in fighting against bacterial infection. Previous studies have shown that P. aeruginosa has the ability to produce and secrete various virulence factors. The ability to cause increased resistance to commonly used antibiotics and the possession of numerous virulence factors render P. aeruginosa a challenging pathogen to treat in clinical settings. Thus, the need for new drug targets in P. aeruginosa is still high and requires animal models and testing of remedies with potential beneficial effects. Many monosaccharides and polysaccharides have been confirmed to protect cells from various environmental factors. In the present study, we found that LBP has direct benefits on P. aeruginosa infection.P. aeruginosa binds to populate related epithelial cell cultures rapidly. Moreover, P. aeruginosa cells were more uniformly distributed on the epithelial cell surface  has a cellular composition that prevents infection from inhaled particles. The ability of bacteria to adhere to mucosal epithelial cells, particularly concerning the respiratory system, is crucial to colonization and infection. In this study, our findings show that  surface . As the  surface . Inhibit surface . SolubleIn this study, PCN-induced apoptosis emanates from two reasons. First, PCN contributed to the expression of pro-apoptotic protein caspase3 and Cyt-c and was elevated to decrease the anti-apoptotic protein Bcl-2 and Bcl-xl expressions. The regulatory mechanisms of apoptosis include but are not limited to caspases and BCL-2 family proteins. Caspase3 is the central molecule in apoptosis. Its activation is regulated by a series of signal transduction cascades, among which the interaction between antiapoptotic Bcl-2 and Bcl-xl proteins plays a vital role. The downregulation of Bcl-2 is reported to release cytochrome c from the mitochondria, resulting in caspase3 activation. Therefore, we speculate that PCN induces the expression of caspase3 by suppressing the expression of anti-apoptotic proteins Bcl-2 and Bcl-xl. Meanwhile, PCN could promote the expression of Cyt-c, which activates caspase9 and the downstream signal caspase3, resulting in the mitochondrial apoptotic cascade. As a result, the cleavage of caspase3 results in the occurrence of apoptosis. This is further substantiated by the data presented in P.A or PCN could cause tissue damage, inflammation, or lesions. For in vivo study, P.A or PCN was systemically administered to mice. Our data show that P.A and PCN infection induced apoptosis protein caspase3 and Cyt-c expression and activation in mice lung and spleen tissues. Specifically, tissue damage is associated with the formation of inflammatory cell infiltration. Excess generation of pro-inflammatory cytokines can cause severe cellular and tissue injury by inducing oxidative damage of biological macromolecules and inflammatory cell infiltration. Cytokine(s) release from pulmonary tissue may trigger lymphocyte apoptosis in the spleen. Our study’s interesting finding is that LBP protects mice lung and spleen from P.A- and PCN-induced injury. Also, LBP could reduce the expression of caspase3, inhibit tissue injury in the lungs, and enhance the release of inflammatory cytokines in the spleen. These results suggest that LBP exerts protective effects in vivo and in vitro.Accumulation of mitochondrial-derived ROS activates redox-sensitive transcription factors, facilitating the production of anti-inflammatory or pro-inflammatory factors. They efficiently produce ROS and inflammatory cytokines, IL-1β, TNF, IL-6, and IL-8. Persistent inflammation can promote secondary tissue injury through the excess production of pro-inflammatory factors, such as TNF-α, IL-1β, and IL-6. The elevated levels of pro-inflammatory cytokines, which may cause immuno-mediated damage to the lungs and other organs, result in acute lung injury and, subsequently, multi-organ dysfunction. After the lungs and the draining lymph nodes, the spleen is the third organ involved in the immunity response generation. Therefore, histological analysis of vital organs is important to evaluate whether Pseudomonas aeruginosa infection-induced lung injury using air-liquid culture as an infection model. We confirm that P. aeruginosa-infected bronchial epithelial cells could lead to the occurrence of apoptosis caused by the overexpression of caspase3 in cells and imbalance of Bcl-2 family proteins. Meanwhile, the release of large amounts of ROS disrupts the redox balance in cells and causes oxidative injury in mitochondrial function. Excess generation of ROS and pro-inflammatory cytokines causes severe cellular and tissue injuries by inducing oxidative damage of biological macromolecules and inflammatory cell infiltration. Simultaneously, we confirm that LBP inhibits mouse bronchial epithelial cell apoptosis. Furthermore, LBP further inhibits the release of inflammatory cytokines and tissue injury by inhibiting ROS’ expression. Our research indicates the potential applicability of LBP as an antimicrobial agent.In this study, we investigate the effects of LBP on